Heparin is a highly sulfated glycosaminoglycan (GAG) consisting of repeating disaccharide units. Unfractionated heparin has an average molecular weight of 12-15 kilodaltons (kD). Most heparin prepared commercially for infusion into human patients is isolated from the gut mucosa of pigs in the form termed “unfractionated heparin” (UFH). Fractionation of UFH by various methods results in low molecular weight heparin (LMWH) with 60% of the polysaccharide chains having a molecular weight less than 8 kD. The anticoagulant affect of UFH is mediated by its interaction with anti-thrombin three (ATIII), a serine protease inhibitor of thrombin.
Binding of UFH to anti-thrombin markedly increases inhibition by ATIII of the coagulation proteases, thrombin and Factor Xa. It is estimated that in the United States alone, about one third of hospitalized patients or 12 million patients per year undergoing surgery and other therapeutic procedures are given UFH or LMWH for treatment or prevention of thrombosis. About 1-5% of these patients develop the disorder “heparin-induced thrombocytopenia” (HIT).
HIT is an adverse reaction to heparin, in which affected patients produce platelet-activating antibodies and develop thrombocytopenia. A subset of these individuals experience arterial or venous thrombosis, which in severe cases can be life-threatening. Experts believe that up to 600,000 people per year develop HIT, which is double the number of breast cancer cases diagnosed annually and nearly equal to the number of new cases of angina diagnosed each year in the United States. Because early diagnosis and treatment can reduce morbidity, it is important that a timely and accurate diagnosis of HIT be made. An accurate diagnosis of HIT requires attention to both clinical findings and laboratory test results.
Conventional treatment for patients suspected of having HIT includes the immediate cessation of all heparin followed by prompt administration of a non-heparin, alternative anticoagulant such as a direct thrombin inhibitor. Such treatments involve additional hospitalization, a considerable expense, and a risk of severe bleeding of 1% per day. Thus, an accurate diagnosis of HIT and characterization of thrombosis risk is critical for effective patient management.
Platelet factor 4 (PF4) is a 32 kD tetrameric protein consisting of four identical 70-amino acid subunits that is released from the alpha-granules of activated platelets and binds with high affinity to heparin, leading to generation of the PF4 epitopes that HIT antibodies recognize. One physiologic role of PF4 may be neutralization of heparin-like molecules on the endothelial surface of blood vessels, thereby promoting coagulation. Other roles appear to include pro-inflammatory activity, inhibition of angiogenesis, and inhibition of megakaryocyte proliferation and platelet production.
HIT is caused by antibodies that recognize PF4 in a complex with heparin. Among available tests for HIT antibody detection, the serotonin release assay (SRA) is considered by many to correlate best with a clinical picture typical of HIT and is often used as a surrogate for HIT diagnosis. However, the SRA is performed routinely only in a few specialized laboratories because of challenges that include the use of radioactivity, labor intensiveness, reliance on fresh platelets, and technical demands of the assay. An alternative, widely used diagnostic test, the PF4-based ELISA, is technically simple and highly sensitive for antibody detection but lacks the necessary specificity to diagnose clinical HIT.
Other recently developed HIT assays show promise but are still investigational and not yet validated for clinical use. It remains unclear why some Heparin-PF4 directed antibodies cause HIT but many others do not. It is generally agreed, however, that antibodies testing positive in the SRA are most likely to be pathogenic and to cause thrombocytopenia as well as thrombosis, the most serious complication of HIT.
Accordingly, there is a need for assays that distinguish between antibodies that activate platelets and those that do not and can be performed in a relatively short time by a typical hospital laboratory.